Method for detection of bromine in urine using liquid chemistry, dry chemistry test pads. and lateral flow

ABSTRACT

This invention is in the field of toxicology and clinical diagnostics. More specifically, this invention provides a single dry chemistry, liquid chemistry, or lateral flow dry chemistry combination test device for use in the detection of adulteration by the addition of bromine(s) to a specimen submitted for Drugs of Abuse (DAU) testing and clinical diagnostic purposes in aqueous fluids, including urine, saliva, serum, blood, sweat extracts, and liquid homogenates of hair.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a division of application Ser. No. 09/556,395, Filed Apr.24, 2000, now U.S. Pat. No. 6,537,823 granted Mar. 25, 2003.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] As the use of illicit drugs in this country has increased, publicconcern over the problems associated with its effects has grown into amajor concern. This concern has led to workplace drug testing in orderto identify, treat, and remove active drug users from the workforce.This trend started in the military, and spread rapidly to lawenforcement and any “safety-sensitive” private sector jobs such asairline pilots, truck drivers, and active crew members of publictransportation. These initial strides into drug testing in the workplacerevealed the obtrusive incursion of drug use and abuse in the dailylives of a significant portion of Americans. Further research indicatedthe staggering costs to public and private industry in terms of lostproductivity, increased health care costs, and human suffering and deathdue to this scourge of drug abuse. As a result, drug testing has rapidlyspread to all areas of the public and private sector. The vast majorityof workplace drug testing has taken the form of urine testing, becauseof ease of collection, low cost, and effective indication of recent druguse. Other forms of testing include analysis of blood, saliva, sweat,and hair.

[0004] Because the effects of a positive test on the individual can besignificant, and traumatic, the analysis procedures must guaranteeaccuracy with the emphasis on zero false positive results. On the otherhand, all efforts must be made to detect all drug users in order toinsure the success of this policy. These two requirements dictate apolicy of close and vigorous scrutiny of the collection, testing, andreporting procedures. Juxtaposed to these closely monitored proceduresis the deep and abiding desire of illicit drug users to avoid detectionin order to keep their use secret, and to keep their jobs. Thus drivenby these key desires, the ingenuity of a few in the drug abusesubculture has led to a plethora of ways to defeat the workplace drugtesting procedures. These “adulteration” methods all conspire to producethe same desired effect: a false negative result which will protect thedrug user's secret.

[0005] Adulteration techniques can be divided into two distinct types.The first utilizes an “in vivo” technique in which the user consumes theadulterant. The second technique utilizes an “in vitro” method in whichthe abuser adds the adulterant directly to the urine specimen submittedfor testing.

[0006] The drug testing procedure involves two distinct parts. Theinitial segment is a panel of screening tests for the individual drugs.If a positive result is obtained in any of these initial tests, then aconfirmation assay is performed for each drug that screened positive.Most adulteration techniques are aimed at the screening process, becauseof the inherent fragile nature of these inexpensive assays which adaptwell to rapid, automated analysis techniques. All screening testsutilize antibody/antigen reactions quantified via an enzyme indicator.On the other hand, confirmation assays are labor and time intensive,highly accurate, expensive, and more difficult to adulterate. Inaddition, the positive screen has already raised a red flag, therebydrawing attention to the sample. The confirmation analysis utilizesGC-MS (gas chromatography mass spectrometry) testing which is consideredthe “gold standard” for drug assays scientifically and legally.

[0007] The “in vivo” methods function in one of three ways. Theseinclude dilution of the analyte of interest to a level below thatrequired for a positive result, decreasing the time required toeliminate the consumed drug, or consuming a compound that will interferewith the screening method. Dilution is effected by consuming a largevolume of liquid together with a diuretic to speed elimination of urine,and a B vitamin to add yellow color to the urine sample. Some commercialin vivo dilution products or “flushes” are sold under the followingnames: Carbo Clean, Test Pure, Kleen Test, Quick Flush, Naturally Klean,Test Free, UA Flush, Zydot's Special Blend, Daily Pure, Vale's QuickClean, Test'n, and UR'n Kleen. Decreasing the elimination time willoften enable the weekend drug user to avoid testing positive on a Mondaymorning drug test. This is accomplished by consuming acidic liquids(e.g. acidic fruit juices or ammonium chloride) to speed up eliminationof basic drugs, or consuming basic liquids to speed up elimination ofacidic drugs. Examples of an internally ingested substance which willdisrupt the screening test procedure include aspirin and mefenamic acid,a prescription analgesic pain killer.

[0008] In vitro methods utilize literally hundreds of products andcompounds that will adversely affect either the screening orconfirmation process. Products affecting the screening process includemany household products (i.e. all types of cleaners including hand,clothes and dishwashing detergents and soaps, table salt, hydrogenperoxide (oxidant), oxidants (such as sodium nitrite, sodium bromate,potassium bromate, bromine, bleach (sodium hypochlorite (C1), anoxidant), fingernail polish remover, vinegar, Drano, liquid plumber,sodium bicarbonate, Visine, fingernail polish, swimming pool cleaningchemicals and acid), or specialty products sold commercially asadulterants (i.e. Urine Luck (contains the oxidizer pyridiniumchlorochromate), Purafyzit, Urine Sured, and THC Free are acid-basedproducts with some including other ingredients such as chromates andnitrites (oxidants), UrinAid and Clear Choice are glutaraldehydecontaining products, Amber-13 contains sulfides, Mary Jane Super Clean13 is a soap, Stealth, and Toxiclean). Commercial products aimed atinterfering with the confirmation process include nitrite (oxidant)containing products Klear and Whizzies and bromine molecule containingproduct known as Stealth.

[0009] Substitution, or using a clean urine sample supplied by a thirdparty, can be either in vivo or in vitro adulteration. In its simplestform, participants hide a clean urine in their clothing and put it intothe specimen collection container (in vitro). Individuals requiring morestealth including those giving observed collections (military andcorrections primarily) may substitute via the in vivo technique whichrequires putting the clean urine into the subject's bladder using acatheter.

[0010] Illicit drug users have learned to falsify urine screening testsby in vitro adulteration of urine samples by the addition of severalreadily available agents, including household products (soap, bleach,etc. . . . ), hydrogen peroxide, and commercially available adulterationproducts, such as “UrinAid and Clear Choice” (glutaraldehyde containingadulterants) or “Urine Luck” (a chromate containing adulterant).

[0011] The vast majority of urine collections are not observed due toprivacy issues. Collection facilities try to prevent in vitroadulteration or substitution by recording the temperature of the sampleas soon as it is collected. It must fall inside the very narrow range of90.5 to 99.8 degrees Fahrenheit. They also may require subjects to leaveexcessive clothing out of the collection room, and provide no hot waterwhich prevents dilution of the sample with water. Obviously, however, itis very easy to secret small quantities of adulterating substances intothe collection room. As little as a pinch of salt or a drop or two ofglutaraldehyde, pyridinium chlorochromate or acid will affect most testscreens. Because the effective amounts of most adulterants are verysmall, even observed collection as required by the military and criminaljustice system can be defeated using the in vitro technique.

[0012] On the other hand, collection facilities currently have noweapons to detect in vivo adulterants, because they are consumed by thedrug user several hours or days prior to collection of the sample.Currently only certain forms of adulteration as already mentioned can bedetected in the laboratory.

[0013] All screening assays can be adulterated. These assays fall intothree types of methods: florescent polarization immunoassay (FPIA),radioimmunoassay (RIA), and enzyme immunoassay (EIA, CEDIA, KIMS:Antibody/agglutination). Toxicology and clinical chemistry literatureincludes numerous studies on the effects of adulterants on the screeningtechnologies and the recommendation to test for their presence in urinesamples. References include Mikkelsen and Ash, “Adulterants CausingFalse Negatives In Illicit Drug Testing”, Clin. Chem. 34/11, 2333-2336(1988); and Warner, “Interference of Common Household Chemicals InImmunoassay Methods For Drugs Of Abuse”, Clin. Chem., 35/4, 648-651(1989).

[0014] Accordingly, a need exists for providing an easy and convenientmanner by which to make a determination of the presence of brominecontaining (oxidative active) adulterants in urine samples which arebeing tested for drugs of abuse. Specifically an assay capable ofdetecting bromine. A further need exists for a convenient manner bywhich such determinations may be made by using rapid analysis manualtechniques (such as a dipstick or lateral flow devices) and automatedtechniques that will advance the art significantly. And, the mostimportant needs is for a device that would detect the adulterant bromineusing just a single assay. This would be a marked advancement in the artand would result in the savings of millions of dollars to the drugtesting laboratories required to perform adulteration testing andobviously this savings would be passed on to the end user (thebusinesses which initially request drug's of abuse assays on perspectiveand current employees). Other advancements that would be made with adevice that is capable of the detection of different forms of bromine inurine in the form of sodium bromate, potassium bromate, or any compoundscontaining bromine that could be introduced into a urine in an effort tointerfere with drugs-of-abuse testing. This is significant deterrent fora person trying to adulterate a urine sample with a bromine containingcompound.

[0015] 2. Description of the Related Art

[0016] This invention is in the field of toxicology. More specifically,this invention provides test strips (i.e. dry chemistry dipsticks, oron-site test modules utilizing thin layer chromatography in a lateralflow format, or other similar technology to the test strip) and liquidchemistry reagents for use in the detection of bromine with a singleassay to include but not limited to sodium bromate, potassium bromate,bromine or analogs of these oxidants or other compounds containingbromine which are capable of producing an oxidative reaction in anaqueous solution which causes adulteration of immunoassays designed todetect Drugs of Abuse (DAU) in aqueous fluids to include but not limitedto urine, saliva, serum, blood, sweat extracts, and liquid homogenatesof hair.

[0017] Currently, all adulteration detection assays actually analyze thetest sample itself for physical or chemical abnormalities. For instance,most of the adulteration products noted above can be identified bydetermining the pH, or specific gravity of the sample. These two assayswill detect the presence of acids, bases, table salt, and high ionicstrength soaps and detergents. It is important to note that there are nodry chemistry (dipstick or lateral flow technology) commercial testsavailable for oxidation-based adulterants (i.e. bromine, in the form ofsodium bromate, potassium bromate, or other analog of bromine (which iscontained in the commercially known product Stealth)). It is importantto note that oxidative activity from an adulterant causes interferencewith the drugs-of-abuse immunoassays as known in the art as well as theextraction procedures used to extract drugs from urine. For example,lets say an employer suspects that an employee is under the influence ofdrugs while performing a sensitive job such as piloting a commercialjet, this individual (who has been smoking cannabinoids) is ordered tosubmit a urine for drug screening and adulterates his or her urine withnitrite. The urine which contains THC (cannabinoids) will be positive onthe initial immunoassay screen. Because oxidants do not interfere withthe initial screening process such as EIA (enzyme immunoassay) which iscommonly used. The lab running the immunoassay drug screen will thenforward the positive sample for confirmation by GC-MS (gaschromatography mass spectrometry). However, during the GC-MS extractionprocedure the oxidant will interfere with the extraction process andprevent any THC from extraction from the urine for confirmation. Thetechnician will run the sample on the GC-MS and the result will benegative for THC. The drug testing lab will have to report out anegative for the drug screen. This has obvious multiple negative effectson the industry. First, the drug abusers gets away with tampering withthe sample, the lab runs the expensive procedure for confirmation byGC-MS and cannot report a positive and thus will get no revenue forrunning the confirmation procedure, and the employer who ordered thedrug screen has an employee who is still under the influence and on thejob, and lastly, the customers on the commercial jet are in for aninteresting if not fatal ride. This is just one of many scenarios thatcould result from the use of oxidants for urine adulteration. It is alsovery important to note that there is no prior art that offers thecapability of a single assay that detects bromine, more specifically inthe form of bromine type oxidant.

[0018] The novel invention described herein describes a method todetermine the presence or absence of bromine in urine or other fluids byliquid and dry chemistry test means which has not been taught prior tothe present art. It should be noted that bromine is not normally foundin urine.

[0019] The only published and patented chemical analysis methods foradulteration testing are liquid assays (aqueous not dry chemistryassays) for pH, specific gravity, glutaraldehyde and nitrite.

[0020] This new invention will clearly indicate if bromine is present ina sample of urine which will cause a false negative or false positiveresult depending upon the type of immunoassay used in the DAU screeningprocess. Currently, the adulteration underground network produces two tofive new methods to fool the drug tests each year. As a result drugtesting facilities are forced to maintain a constant vigil for anyunusual results. It is well known that adulteration of samples submittedfor DAU testing cost the drug testing laboratories, employers, taxpayersand everyone involved in the drug screening process countless millionsof dollars every year.

[0021] A thorough search of patents and research revealed no relativeart (i.e., prior art) with any correlation to this technology. The artof testing for adulteration in urine as previously delineated in theliterature describe various techniques including manual methods formeasuring pH (meter or pH papers), specific gravity (via refractometeror dipstick), temperature, and liquid chemical analysis for specificadulterants such as glutaraldehyde (as described by U.S. Pat. No.5,464,775). No reference, however, has described this new art asdelineated here. The previous art will be enumerated here to furtherillustrate the unique advancement in the field of adulteration detectionthe present device yields. Other than U.S. Pat. No. 5,464,775 (whichdoes not mention or teach any of the present art's indicators, or testmeans for the detection of adulterants containing bromine), none of thefollowing patents teach the use of urine as the test subject ofadulteration detection. It has been acknowledged in the art that randomurinary sample matrices are very complex, and consist of many urinaryconstituents which create strong buffering and interference problems(e.g. cannibal like enzymes such as protease). In addition, diseasestates will significantly impact the nature of urinary contents. Urineis also the repository of all of the body's waste products includingexcess parent nutrients, vitamins, drugs, and their metabolites. Thesewaste chemicals vary from person to person and significantly contributeto the individual uniqueness that makes assay design for urinaryconstituents more difficult than any other body fluid. All of thesefactors impact an assay's ability to obtain acceptable precision andaccuracy. The ability of an assay to analyze a biological liquid such assaliva, therefore, rarely ever translates to an effective assay forurine. Therefore the present invention's ability to effectively copewith the random urine sample makes it unique.

[0022] Another patent, U.S. Pat. No. 3,603,957, discloses the use ofassay test strips, but fails to teach a method for the determination ofadulteration of a test sample submitted for drugs of abuse testing. Italso doesn't teach a method to determine the presence or absence of anysubstance or adulterant which interferes with the drug's of abuse testassay such as oxidants. The patent doesn't teach the use of the presentart's reaction formula to dry chemistry format called a dipstick orlateral flow technology that not only is completely novel, but preventscross contamination between test pads typically found on a test strip(dipstick). In addition, this patent also failed to mention any methodsfor determination of adulteration of samples submitted for analysis ofdrugs of abuse by dipstick, lateral flow, calorimetric, liquid reagent(automated) or other suitable means.

[0023] Another patent, U.S. Pat. No. 4,301,115, discloses the use ofassay test strips, and the ability of the assay strips to resist crosscontamination between reactant areas (chemically impregnated test pads),but fails to teach a method for the determination of adulteration of atest sample submitted for drugs of abuse testing. The patent doesn'tteach the use of the dry chemistry format utilizing either a dipstick orlateral flow device, liquid reagent (automated) method or mention anymethods for determination of adulteration.

[0024] Another patent, U.S. Pat. No. 5,447,837, does mention the use ofassay test strips but again fails to disclose a method for thedetermination of adulteration. This is a method for detection of anantigenic substance in human, biological samples. This patent also failsto mention the use of a reaction formula that is adaptable to the drychemistry format utilizing either a dipstick or lateral flow device. Italso doesn't teach a method to determine the presence or absence of anysubstance or adulterant which interferes with said reaction. Inaddition, this patent also failed to mention any methods fordetermination of adulteration of samples submitted for analysis of drugsof abuse by dry chemistry, liquid chemistry, calorimetric, or othersuitable means.

[0025] Published literature and the prior art describes techniques suchas ELISA that have been used to determine the presence of drugs ofabuse, but these technologies have no relevant bearing on the presentdevice. Previously taught technologies include measurement of pH (meteror pH papers), specific gravity (via refractometer or dipstick),temperature, and liquid chemical analysis for specific adulterants suchas glutaraldehyde. Therefore, in a nutshell, the present device providesan absolute novel approach to adulteration testing and lateral flowtesting using dry chemistry test pads and automated liquid reagenttesting.

[0026] GC-MS, a confirmation, assay, is performed to verify the urinesthat screen positive for drugs of abuse. The GC-MS analysis costs 100times as much as the initial screen ($100 vs $1). Every additionalunnecessary GC-MS performed drives up the overall cost of drug testing.Eliminating these additional, unnecessary assays will save millions ofdollars per year. False positive drug screens also strongly impacton-site testing. In most situations utilizing on-site tests (on sitedevices such as dipstick or lateral flow devices require noinstrumentation, making these devices ideal for collection and on sitefacilities) the employee is screened upon arrival for work. If apositive is obtained using the on-site test, a second sample isforwarded to the lab for GC-MS confirmation and the employee issuspended from work or reassigned to other duties until the results ofthe test are known. Therefore, it is of vital importance that theemployer and laboratory know if the sample has had an adulterant added,to save time, money, and possibly lives.

[0027] Not surprisingly, it is known and is illustrated here that agreat need exists in the field of workplace drug testing for rapid,economical, and effective adulteration analysis of samples submitted fortesting, whether liquid chemistry and/or dry chemistry methodology usingdipsticks or lateral flow test devices (for single use and for on-sitecollections). The present invention does detect oxidants in the form ofbromine effectively with a single assay and therefore and accordingly,the present device provides an easy and convenient manner by which tomake a determination of the presence or absence of bromine oxidizingadulterants in samples submitted for drugs of abuse testing. The presentart's use of lateral flow also enables the removal of any interferenceof any cross over of reagents or fluid from one test pad to anotherwhich is one of the exclusive problems with dipsticks.

[0028] It is clear that a need exists for a convenient manner by which adetermination of intentional adulteration by the use of bromine (oranalogs of bromine) oxidants can be made utilizing a rapid automatedanalysis utilizing a liquid reagent format of the present device ormanual analysis in the form of dry chemistry (dipstick) or lateral flowtest devices. These and other advances in the current state of the artwill become evident in view of the present specification and claims.

SUMMARY OF THE INVENTION

[0029] Briefly stated, the present invention relates to the test devicesfor detecting the presence of an adulterant containing bromine or thepresence of bromine for clinical purposes in a liquid test sample andthe methods for making said devices. This invention is in the field oftoxicology and clinical diagnostics. More specifically, this inventionprovides dry chemistry test strips (i.e. dipsticks, or dry chemistry andlateral flow [thin layer chromatography] test means) or automated ormanual liquid reagent means for use in the detection of adulteration ofhuman, biological samples (e.g. urine, blood, serum, saliva, sweatextracts, and hair homogenates) to be tested for DAU's via immunoassays.This invention achieves this goal by measuring the absence or presenceof oxidants in the form of bromine (or analogs of bromine) in a testsample. And, this invention provides a unique method for preventingcross contamination between test pads (reactant areas) on dipsticks bythe present inventions use of the dipstick test pad and lateral flowdevice technology. This invention provides a previously unavailable drychemistry or liquid chemistry method for determining adulteration of atest sample by measuring the presence of bromine as required currentlyby the Department of Transportation, Nuclear Regulatory Commission,College of American Pathologist's Forensic Urine Drug Testing program,and the U.S. Department of Health and Human Service's SAMSHA program onall urines assayed for drugs of abuse under their protocols. And, thisinvention also provides a test means to detect bromine in a urine orother biological samples for clinical diagnostic purposes.

[0030] The present invention encompasses a method that can utilizeseveral different techniques. The techniques would employ the manualmethod using dry chemistry dipsticks and the method of combining drychemistry dipstick reactant areas (test pads) with lateral flow thinlayer chromatography or the method of using a liquid reagent that iscompatible with automated analyzers that provide high speed quantitativeanalysis which would be much less labor intensive than the manualmethods providing a savings in time and money. For example, SAMSHA hasmade adulteration testing mandatory and the present art provides a novelmethod for performing adulteration testing for bromine containingoxidants (to include but not limited to sodium bromate, potassiumbromate, bromate or bromine analogs, and Stealth) with or without theuse of or need for instrumentation, thus allowing for the laboratory tosuspend any further testing of a specimen failing an adulteration check.This course of action reduces technician time, and provides asignificant economic savings. The widespread utility of the present artalso provides the drug testing laboratory, collection site (where theurine is actually collected), or other users the choice of using the drychemistry (manual) or automated liquid means which ever method bestsuits their situation or needs.

[0031] The present arts technique utilizes two dry chemistry techniques,one is dipsticks, which is a carrier dependent, rapid test that usesabsorbent medium such as paper which have been impregnated with achemical formulation to detect adulteration. After dipping one(dipstick) into a liquid test sample, a reaction takes place. Saidresulting reaction will yield a color change indicating a positive ornegative result (i.e. presence or absence of chromate, an adulterant).The other technique is the use of lateral flow in combination with a drychemical test pad. The lateral flow device is a rapid test that usesabsorbent medium such as paper which has been impregnated with thechemical formulation to detect adulteration. The paper, afterimpregnation, is then placed on a lateral flow medium, such asnitrocellulose paper, glass fiber paper, or other suitable wickingmaterial that will deliver the test sample to the impregnated paper. Thelateral flow device works by dipping one end of the lateral flow device(LFD) into a sample (urine for example). The urine migrates up (along)the paper (or absorbent material) to the reactive sites (test paper)containing reagents (reactive ingredients). The urine constituents reactwith the assay reagents during the migration process and yield visibleresults. The urine can also be droppered onto the LFD and the samplewill then migrate along the paper.

[0032] The ease of use and rapid results obtained by the present art'smethodology illustrate the unique utility of this testing technique. Inaddition, very little technical expertise is required to perform thistype of assay (no instrumentation required). Furthermore, the earlydetection of adulteration (prior to DAU screening) facilitates fasterreplacement with a fresh, untainted specimen from the participant,yielding more accurate information. This novel concept for adulterationmonitoring provides an enormous savings of time and money. Adulterationtesting utilizing these techniques are currently not available and havenever been taught.

[0033] An important aspect of adulteration testing in urine is thesensitivity of the test method. Both techniques taught here have aneffective sensitivity range comparable to the target immunoassay. Thedipstick test and LFD dipstick hybrid (to be known as the LFD hybrid)have a quantitative to qualitative assay range. The results areevaluated via one of two categories: negative and positive.

[0034] The present arts technique also utilizes a liquid chemistry testmeans that allows for rapid analysis via an automated analyzer that canyield high speed quantitative results. This will result in rapid testresults, improved accuracy, lowered labor cost, and better turn aroundon a high volume of test. This automated method is only limited by thespeed of the automated analyzer. Some analyzers currently on the marketare capable of over 10,000 test per a hour. The ability of the presentart to perform a single assay on a high speed automated analyzer that iscapable of detecting bromine or bromine containing adulterants has neverbeen present or taught in the prior art.

[0035] It is currently known in the art that enzyme and antigen/antibodyreaction kinetics are related to the rate of change in analytical,biological systems. The variables that affect this rate of changeinclude concentration of reactants and product, temperature, pH, ionicstrength, buffer strength, and other parameters. Many commercial andhousehold adulterants dramatically affect the parameters noted above.The present art's innate and unique ability enables it to determine thepresence of bromine (Stealth) in urine. As it is known in the art urineis a very complex matrix and the measurement of bromine in urine has totake into account many factors which will affect the assay.

[0036] The composition of the formulation to be applied to the drychemistry dipstick, LFD hybrid and liquid chemistry method are composedof indicator(s) (visible calorimetric), and buffer(s).

[0037] Briefly stated, the present invention relates to test devices formeasurement of bromine(s) in urine but could also work in other matricessuch as blood, saliva, or other fluids that come from the human body andthe procedures for making said test means. This invention is in thefield of clinical diagnostics. More specifically, this inventionprovides dry chemistry dipsticks (DCD's or on-site test modules), thinlayer lateral flow chromatographic dry chemistry technology (LFD's), andthe combination of both in a unique hybrid that is not known prior tothe present art and liquid chemistry reagents for automated and manualuse. That is to say (in it's simplest terms) that this unique hybrid(LFD) will encompass the use of a dry chemistry test pad resting on thesurface of a wicking material (such as nitrocellulose) acting as a fluiddelivery device. This new art can utilize aqueous, biological specimensincluding urine, saliva, sweat extracts, blood, and serum. Thus, thisinvention provides a unique method for bromine (oxidant) measurementutilizing rapid test devices including the automated method as well asthe DCD, and LFD methodology thereby enabling in-home testing throughover-the-counter (OTC) sales. This is an enormous advancement in theart. These advances and improvements of the present device over theprior art provides the health care and drug testing industry withpowerful new clinical and diagnostic tools.

[0038] A thorough search of the literature reveals no relative artresembling this technology; therefore, this invention is clearly a novelcreation, and is not obvious to anyone skilled in the art of toxicologyand clinical chemistry.

DETAILED DESCRIPTION OF THE INVENTION

[0039] This instant invention is a single assay in the form of a liquidchemistry reagent, dry chemistry dipstick or lateral flow device inconjunction with using a dry chemistry test pad for the detection ofoxidants in the form of bromine or analogs of bromine (or other statesof bromine such as sodium bromate, potassium bromate, etc.) in samplematrices consisting of urine or other biological specimens (e.g. saliva,serum, blood, sweat extracts, and hair homogenates). The bromineadulteration detection assay that makes up the instant invention maytake the form of dry chemistry dipsticks or dry chemistry test padlateral flow hybrid, both of which are composed of some or all of thefollowing compounds: buffer(s) and color indicator(s), hereinafterreferred to as the adulteration reagent or the liquid automated reagentdesigned for high speed automated analyzers also composed of some or allof the following compounds: buffer(s) and color indicator(s). It can benoted that the liquid reagent method could also be used manuallyemploying spectrophotometers or other types of visual detectiontechnology. Buffering of the reactants is critical to the adulterationreagent, because pH plays a vital role in the reaction kinetics. In thecase of the dipstick and the dipstick/lateral flow hybrid (which can beknown as the “DLFH” device), bromine adulteration reagent components areimpregnated on the test strip pad composed of solid, absorbentcarrier(s), usually known as test pads. In the case of dipsticks, thesetest pads are typically affixed to a solid support (usually plastic).This device is then submerged in the liquid test sample, removed, and ameasurable (i.e. visible) response is observed. Or in the case of theDLFH, the dry chemistry test pad is chemically impregnated identicallyto the dipstick. The test pad is then placed in fluid (direct) contactwith lateral flow paper (such as nitrocellulose). This device is thenexposed to a fluid (urine for example). The urine then migrates to thelocation of the test pad, saturates the test pad, and the reaction takesplace.

[0040] The adulteration reagents of the device constitute the heart ofthe analytical response provided by it, and is comprised of one or morereagent compositions responsive to any number of chemical componentscapable of adulterating a test sample. The reagents, in the broadestsense produces a detectable manifestation of the presence of saidchemical component(s); said chemical components being capable ofadulterating a test sample thereby yielding an incorrect result(interfering with) for the target DAU immunoassay. The response can bein the form of the appearance or disappearance of a color, or thechanging of one color to another. Said measurable response may also beevidenced by a change in the amount of light reflected or absorbedduring the reaction of interest. The analytical arts are replete withexamples of these types of detectable responses.

[0041] In the present invention, there is provided a dry chemistry teststrip in the form of a dipstick or DLFH for the detection ofadulteration in urine (or other biological fluids including saliva,sweat extracts, serum, blood, and hair homogenates) comprising a solid,carrier matrix in the form of a dry chemistry dipstick containing anindicator compound and buffer.

[0042] The present technology does not predict or forecast the obviousadvancement in the art to encompass the present invention, nor does ithint at the extraordinary improvement the present invention provides inthe field of adulteration detection. While urine is the sample matrix ofchoice for this instant invention (and for the immunoassays currently ingeneral use) it is well within the scope of this novel invention toutilize it in the analysis of other sample matrices including saliva,sweat extracts, serum, hair homogenates, gastric contents, cerebralspinal fluid, and blood. While the assay is designed for adulterationdetection, the assay could be used in other fields for other uses suchas clinical diagnostics, manufacturing, or research.

[0043] The remarkable discovery of the new art formula will require thepresence of an indicator(s) for bromine in urine (as well as the othermatrices mentioned) that was unknown prior to this art. The newlydiscovered and suitable indicators for the present art formula are;1,2,3,4-tetrahydrobenzo(h)quinolin-3-ol,1,2,3,4-tetrahydrobenzo(h)quinolone,1,2,3,4-tetrahydrobenzo(h)quinaldine,3-hydroxy-1,2,3,4-tetrahydrobenzo(h)quinolone,3-hydroxy-N-methyl-1,2,3,4-tetrahydrobenzo(h)quinolone,3-acetoxy-N-methyl-1,2,3,4-tetrahydrobenzo(h)quinolone,N-methyl-1,2,3,4-tetrahydrobenzo(h)quinolone, 1,3-phenylenediamine,1,2,3,4-tetrahydroquinoline, 1,2,3,4-tetrahydroisoquinolinehydrochloride, 7,8-benzoquinoline,1,2,3,4-tetrahydro-3-isoquinolinecarboxylicacid hydrochloride,1,2,3,4-tetrahydro-1-napthylamine hydrochloride, napthylamine,N,N-dialkyl-alpha-napthylamine, phenolphthalin,2,2′-Azino-di-(3-ethylbenzthiazolinesulfonic acid),2,2′-Azino-di-(3-ethylbenzthiazolinesulfonic acid) diammonium salt,cyanoditoly tetrazolium chloride, 3,3′-diaminobenzidine, o-dianisidine,dimethoxybenzidine, 0-phenylenediamine, 3-amino-9-ethylcarbazole,3,3′-5,5′-tetramethylbenzidine, dimethoxybenzidine, 8-hydroxyquinoline,m-phenylenediamine, 3-dimethylaminobenzoic acid, 5-aminosalicylic acid,and 4-chloro-1-napthol, and another indicator that is suitable for thepresent device is 4-aminoantipyrine in combination with one of thefollowing compounds; p-hydroxybenzene sulfonate, p-hydroxybenzoic acid,n-ethyl-n-(2-hydroxy-3-sulfopropyl)-m-toluidine,n-ethyl-n-sulfopropyl-m-toluidine, 2-hydroxy-3,5-dichlorobenzenesulfonicacid, 3-hydroxy-2,4,6-triiodobenzoic acid, and3-hydroxy-2,4,6-tribromobenzoic acid. In addition, some of the aboveindicators require a diazotizable amine in the form of sulfanilic acid,arsanilic acid, sulfanilamide, aminobenzoic acid, or other suitableamine. It is understood that the present arts discovery of the use ofindicators such as the ones mentioned above or others that have not beenmentioned that are sensitive to bromine oxidation-reduction reactions inbiological matrices such as urine and are capable of producing adetectable response in the presence of a bromine oxidation-reductionreaction are within the present art. Therefore the use of a bromineoxidation-reduction indicator that is not mentioned here would fallwithin the spirit and scope of the present invention.

[0044] This new art formula will require appropriate buffering. Suitablebuffers may include any of the following (referred to here by theircommonly used acronyms): citrate, borate, borax, sodium tetraboratedecahydrate, sodium perchlorate, sodium chlorate, sodium carbonate,(Tris[hydroxymethyl]aminomethane), MES (2-[N-Morpholino]ethanesulfonicacid), BIS-TRIS(bis[2-Hydroxyethyl]iminotris[hydroxymethyl]methane;2-bis[2-Hydroxyethyl]amino-2-[hydroxymethyl-1,3-propanediol),ADA (N-[2-Acetamidol]-2-iminodiacetic acid;N-[Carbaoylmethyl]iminodiacetc acid), ACES(2-[(2-Amino-2-oxoethyl)amino]ethanesulfonic acid;N-[2-Acetamido]-2-aminoethanesulfonic acid), PIPES(PiperazineN-N′-bis[2-ethanesulfonic acid)]; 1,4-Piperzinedethanesulfoicacid), MOPSO (3-[N-Morpholinol]-2-hydroxypropanesulfonic acid), BIS-TRISPROPANE (1,3-bis[tris(Hydroxymethyl)methylamino]propane), BES(N,N-bis[2-Hydroxyethyl]-2-aminoethaesulfonic acid;2-bis(2-Hydroxyethyl)amino]ethanesulfonic acid), MOPS(3-[N-Morpholino]propanesulfonic acid), TES(N-tris[Hydroxymethyl]methyl-2-aminomethanesulfonic acid; 2[2-Hysroxy-1,1-bis(hydroxymethyl)-ethyl]amino)ethanesulfonic acid),HEPES (N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]), DIPSO(3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesulfonic acid), TAPSO(3-[N-tris(Hydroxyethyl)methylamino]-2-hydroxypropanesulfonic acid),HEPPSO (N-[2-Hydroxythyl]piperazine-N′-[2Hydroxypropanesulfonic acid]),POPSO (Piperazine-N,N′-bis[2-hydroxypropanesulfonic acid]), EPPS(N-[2-Hydroxyethyl]piperazine-N′-[3-propanesulfonic acid), TEA(triethanolamine), TRICINE (N-tris[Hydroxymethyl]methyllycine;N-[2-Hydroxy-1-1-bis(hydroxymethyl)etyyl]glycine), BICINE(N,N-bis[2-Hydroxyethyl]glycine), TAPS(N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid;([2-Hdroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-propanesulfonic acid),AMPSO (3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonicacid), CHES (2-[N-Cyclohexylamino]ethanesulfonic acid), CAPSO(3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid), AMP2-Amino-2-ethyl-1-propanol, CAPS (3-[cyclohexylamino]-1-propanesulfonicacid), hydrochloric acid, phosphoric acid, succinic acid, lactic acid,sulfuric acid, nitric acid, chromic acid, boric acid, perchloric acid,potassium hydrogen tartrate, potassium hydrogen phthalate, calciumhydroxide, phosphate, bicarbonate, sodium hydroxide, potassiumhydroxide, citric acid, oxalic acid, tartaric acid, oxalate orsuccinate. Other buffers with an effective pK and pH range, and capacitysuitable for maintaining the sample-reagent mixture within the requiredparameters of the assay's reaction mechanism may be added to the abovegroup, however acidic buffers are preferred.

[0045] Manufacture of the dry chemistry dipsticks may require theaddition of thickeners as taught in the art. Some compounds commonlyused for this purpose include: polyvinylpyrrolidone, algin, carragenin,casein, albumin, methyl cellulose, and gelatin. The typical range ofconcentration for these thickeners is about 0.5 to 5.0 g per 100 ml.Wetting agents or surfactants are also typically used in dry chemistry.For dry chemistry applications, wetting agents aid in even distributionof the chemicals and promote even color development. Acceptable wettingagents can be hydrophilic polymers, or cationic, anionic, amphoteric, ornonionic species. Some commonly used wetting agents include sodiumdodecyl-benzene sulphonate, sodium lauryl sulphate, benzalkoniumchloride, N-lauroylsarcosine sodium salt, Brij-35, Tween 20, TritonX-100, dioctyl sodium sulphosuccinate, and polyethylene glycol 6000.Wetting agents can be added to dipstick impregnation solutions inamounts of 0.5% to 5.0%, and 0.1% to 1.0% in liquid reagents.

[0046] Color enhancers may be used such as sucrose, lactose, glucose orother compounds. Color enhancement can be defined as intensificationand/or alteration in some manner the color that is produced by thereaction to improve the measurement of the detectable response.

[0047] The production of dry chemistry test strips for the presentinvention can utilize any form of absorbent, solid phase carrierincluding filter paper, cellulose or synthetic resin fleeces inconjunction with liquid solutions of reagent compositions in volatilesolvents. This can be carried out in one or more impregnation steps.Each impregnation may contain one or more of the chemical compoundsmaking up the assay reagent composition; the exact procedure is dictatedby the inter-reactivity of the assay constituents and the order in whichthey may have to react with the analyte of interest.

[0048] In the case of the DLFH, the lateral flow invention it canutilize any form of absorbent, solid phase carrier that is capable oftransporting a fluid. These can include filter paper, cellulose orsynthetic resins. More specifically, the lateral flow material caninclude cellulose, cellulose acetate, nitrocellulose, mixed ester,teflon, polyvinylidene difluoride (PVDF), polytetrafluoroethylene(PTFE), polysulfone, cotton linter, non-woven rayon, glass fiber, nylon,ion exchange or other suitable membranes or solid support.

[0049] After impregnation, the dipsticks are dried, cut into strips,glued to a support structure (usually a flexible, flat, plastic stick)as part of a “sandwich” composed of the handle, test pad, and asynthetic resin film and/or a fine-mesh material as known in the art. Inaddition, the instant invention may be combined with the water-stablefilm to produce a dipstick in which the excess sample fluid can be wipedoff in order to improve the accuracy and precision of the results.

[0050] The following examples are provided to further illustrate theinventive aspects of the present discovery, and to further exemplifypreferred embodiments. As such, they are intended as merelyillustrative, and are not to be construed as limiting the scope of theclaims appended hereto.

EXAMPLE 1

[0051] This is a method for manufacturing a dry chemistry dipstick (teststrip) with a solid carrier for the detection of bromine in urinesamples.

[0052] Filter paper is successively impregnated with the followingsolutions and dried at 25 degree C.:

[0053] Solution I

[0054] citric acid 21.0 g

[0055] p-arsanilic acid 3.0 g

[0056] 1,2,3,4-tetrahydrobenzo(h)quinolin-3-ol 7.0 g

[0057] add propanol to make 1000 mL total volume of solution

[0058] In this example a dipstick was prepared in accordance with theinstant invention. The device comprised a paper carrier or solid matrixincorporated with the composition of solution 1 above. Note that theconcentrations of any of the following examples can be varied to suitthe dipstick device format (dependent upon paper type, or use ofsemi-permeable membrane or other suitable material). This example iscarried out using the following procedure. To produce the test means, apiece of Whatman 3 MM filter paper having approximate dimensions of 1inch by 3 inches was impregnated with solution 1 by immersing the paperinto solution 1. The paper was then dried by using forced air notexceeding 25° C. The dried paper is then laminated to one side of apiece of double-sided adhesive transfer tape commercially available from3M Company, St. Paul, Minn. 55144. The laminate is then slit intoportions measuring 3 inches by 0.2 inches. One portion is then attached,via the unused adhesive side to a polystyrene sheet measuring about 1.5inches by 3 inches and the resulting laminate is slit parallel to itsshort dimension to form test devices comprising a, 1.5 inch oblongpolystyrene strip carrying a square of the impregnated papers at oneend, the other end serving as the handle. When the dipstick thusobtained is dipped into a urine submitted for drugs of abuse testing,and no uniform blue to purple color develops then no bromine is present.Conversely, if any concentration of bromine are present in the urine ata 0.01% concentration or greater a blue to purple color will developthus confirming the presence of bromine. The color can vary dependentupon a variety of factors such as color of urine, concentration ofurine, indicator used, etc.

[0059] In summary, Example 1 is as follows: the foregoing dry chemistrytest strip (dipstick) method for the detection of bromine in a sample ofurine comprises the steps of preparing a test means by successivelyimpregnating a solid, carrier matrix with reagent solutions, drying theimpregnated, solid test means, then dipping said dried test means intourine, and finally observing any color change in the presence or absenceof bromine.

[0060] The following changes to the above reagent solution will remainwithin the scope and function of this invention and will have similarresults to the example above. The indicator pair in solution 1,p-arsanilic acid and 1,2,3,4-tetrahydrobenzo(h)quinolin-3-ol, could besubstituted with one or more of the following compounds including1,2,3,4-tetrahydrobenzo(h)quinolone,1,2,3,4-tetrahydrobenzo(h)quinaldine,3-hydroxy-1,2,3,4-tetrahydrobenzo(h)quinolone,3-hydroxy-N-methyl-1,2,3,4-tetrahydrobenzo(h)quinolone,3-acetoxy-N-methyl-1,2,3,4-tetrahydrobenzo(h)quinolone,N-methyl-1,2,3,4-tetrahydrobenzo(h)quinolone, 1,3-phenylenediamine,1,2,3,4-tetrahydroquinoline, 1,2,3,4-tetrahydroisoquinolinehydrochloride, 7,8-benzoquinoline,1,2,3,4-tetrahydro-3-isoquinolinecarboxylicacid hydrochloride,1,2,3,4-tetrahydro-1-napthylamine hydrochloride, napthylamine,N,N-dialkyl-alpha-napthylamine, phenolphthalin,2,2′-Azino-di-(3-ethylbenzthiazolinesulfonic acid),2,2′-Azino-di-(3-ethylbenzthiazolinesulfonic acid),2′-Azino-di-(3-ethylbenzthiazolinesulfonic acid) diammonium salt,cyanoditoly tetrazolium chloride, 3,3′-diaminobenzidine, o-dianisidine,dimethoxybenzidine, m-phenylenediamine, 3-amino-9-ethylcarbazole,3,3′-5,5′-tetramethylbenzidine, dimethoxybenzidine, 8-hydroxyquinoline,o-phenylenediamine, 3-dimethylaminobenzoic acid, 5-aminosalicylic acid,and 4-chloro-1-napthol, and another indicator that is suitable for thepresent device is 4-aminoantipyrine in combination with one of thefollowing compounds; p-hydroxybenzene sulfonate, p-hydroxybenzoic acid,n-ethyl-n-(2-hydroxy-3-sulfopropyl)-m-toluidine,n-ethyl-n-sulfopropyl-m-toluidine, 2-hydroxy-3,5-dichlorobenzenesulfonicacid, 3-hydroxy-2,4,6-triiodobenzoic acid, and3-hydroxy-2,4,6-tribromobenzoic acid. In addition, some of the aboveindicators require a diazotizable amine in the form of sulfanilic acid,arsanilic acid, sulfanilamide, aminobenzoic acid, or other suitableamine.

[0061] The citric acid buffer in solution 1, may be substituted with oneor more of the following buffers: borate, borax, sodium tetraboratedecahydrate, sodium perchlorate, sodium chlorate, sodium carbonate, MES(2-[N-Morpholino]ethanesulfonic acid), BIS-TRIS(bis[2-Hydroxyethyl]iminotris[hydroxymethyl]methane;2-bis[2-Hydroxyethyl]amino-2-[hydroxymethyl-1,3-propanediol),ADA (N-[2-Acetamidol]-2-iminodiacetic acid;N-[Carbaoylmethyl]iminodiacetc acid), ACES(2-[(2-Amino-2-oxoethyl)amino]ethanesulfonic acid;N-[2-Acetamido]-2-aminoethanesulfonic acid), PIPES(PiperazineN-N′-bis[2-ethanesulfonic acid)]; 1,4-Piperzinedethanesulfoicacid), MOPSO (3-[N-Morpholinol]-2-hydroxypropanesulfonic acid), BIS-TRISPROPANE (1,3-bis[tris(Hydroxymethyl)methylamino]propane), BES(N,N-bis[2-Hydroxyethyl]-2-aminoethaesulfonic acid;2-bis(2-Hydroxyethyl)amino]ethanesulfonic acid), MOPS(3-[N-Morpholino]propanesulfonic acid), TES(N-tris[Hydroxymethyl]methyl-2-aminomethanesulfonic acid; 2[2-Hysroxy-1,1-bis(hydroxymethyl)-ethyl]amino)ethanesulfonic acid),HEPES (N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]), DIPSO(3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesulfonic acid), TAPSO(3-[N-tris(Hydroxyethyl)methylamino]-2-hydroxypropanesulfonic acid),HEPPSO (N-[2-Hydroxythyl]piperazine-N′-[2Hydroxypropanesulfonic acid]),POPSO (Piperazine-N,N′-bis[2-hydroxypropanesulfonic acid]), EPPS(N-[2-Hydroxyethyl]piperazine-N′-[3-propanesulfonic acid), TEA(triethanolamine), TRICINE (N-tris[Hydroxymethyl]methyllycine;N-[2-Hydroxy-1-1-bis(hydroxymethyl)etyyl]glycine), BICINE(N,N-bis[2-Hydroxyethyl]glycine), TAPS(N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid;([2-Hdroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-propanesulfonic acid),AMPSO (3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonicacid), CHES (2-[N-Cyclohexylamino]ethanesulfonic acid), CAPSO(3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid), AMP2-Amino-2-ethyl-1-propanol, CAPS (3-[cyclohexylamino]-1-propanesulfonicacid), hydrochloric acid, phosphoric acid, lactic acid, sulfuric acid,nitric acid, chromic acid, boric acid, perchloric acid, potassiumhydrogen tartrate, potassium hydrogen phthalate, calcium hydroxide,phosphate, bicarbonate, sodium hydroxide, potassium hydroxide, oxalate,tartaric, or succinate. Or other buffers with an effective pK and pHrange, and capacity suitable for maintaining the sample-reagent mixturewithin the required parameters of the assay's reaction mechanism may beadded to the above group, however acidic buffers are preferred.

EXAMPLE 2

[0062] This is a method for manufacturing a liquid, carrier-free reagentfor the adulteration detection of bromine in urine.

[0063] Prepare a Solution Containing:

[0064] Bromine Solution I (R1)

[0065] HCl (hydrochloric acid concentrate) 21.0 mL

[0066] 1,3-benzenediamine 2.1 g

[0067] distilled water added to 1000 mL total volume of solution

[0068] pH the solution to a value of approximately 2.0

[0069] lab notes: a) Buffer strength is preferably 0.001 Molar orgreater

[0070] Bromate Calibrator Formulations

[0071] Zero (0) Calibrator:

[0072] 1 liter of 0.2 micron filtered normal human urine with noadulterants or drugs present, and 0.01% sodium azide.*

[0073] 5.0% Calibrator:

[0074] 1.0 mg/dl potassium bromate**

[0075] 1.5 g monosodium phosphate***

[0076] 100 mL of 0.2 micron filtered normal human urine with noadulterants or drugs present*

[0077] pH the solution to a value between 3.0 and 11.0

[0078] lab notes:

[0079] *Human urine can be substituted with distilled water, syntheticurine or other suitable solvent. The bacterial inhibitor sodium azidecould be replaced with chloroamphenicol or other suitable bacterialinhibitors that would inhibit the growth of bacteria.

[0080] ** Potassium Bromate could be substituted with any suitableconcentration of oxidant as a calibrator such as bleach, nitrite,hydrogen peroxide (stabilized preferably), bromine, or other oxidants

[0081] *** The monosodium phosphate buffer could be substituted with anysuitable buffer such as the ones mentioned above that would place theliquid solution at an appropriate pH that is suitable with thecalibrator being used (note that these different bromates would requireddifferent pH ranges for stability) and would in addition aid instabilizing the calibrator in solution.

[0082] The reagent system of the instant invention (liquid reagent) isintended for use on any automatic chemistry analyzers with open channelcapability including Olympus AU 5000 series, Hitachi 700 series, andmany others. The reagent as outlined in Example 2 is used in thefollowing manner: the one component of the reagent composition (R-1) isplaced in the reagent compartment of the analyzer; samples, calibrators,and controls are aliquoted into sample cups which are then placed on theanalyzer. An aliquot of 10 uL of each specimen is then pipetted into asingle, discrete cuvette followed by the addition of 125 uL of the firstreagent, R-1, and mixed; A first spectrophotometer reading is then takenfollowed by a second after a specified incubation period (i.e. oneminute for this example) at the specified wavelength (between 340 and800 nm). The spectrophotometer readings are then recorded. In thisinstance the assay is read at 415 nm, 540 nm or 660 nm. The absorbancecan vary from spectrophotometer based on differences in the width ofcuvette, bulb strength, filter used, and detectors used to name a few.The absorbance of samples, and controls are printed and then compared tothe calibrator's absorbance. The quantitative value for bromineconcentration is then calculated. Any concentration of bromine greaterthan 1.0 mg/dL is considered adulterated.

[0083] Please note that in some cases an adulterant or abnormalconstituent in a sample may cause a false positive. This scenario willyield a very significant increase in the cost of analysis, because aGC-MS assay must then be performed to verify the screening positive. TheGC-MS analysis costs 100 times as much as the screen ($100 vs $1). Everyadditional unnecessary GC-MS performed drives up the overall cost ofdrug testing. Eliminating these additional, unnecessary assays will savemillions of dollars per year. False positive drug screens also stronglyimpact on-site testing. In most situations utilizing on-site tests, theemployee is screened on arrival for work. If a positive is obtainedusing the on-site test, a second sample is forwarded to the lab forGC-MS confirmation and the employee is suspended from work or reassignedto other duties until the GC-MS results are known. If the positive is afalse one due to interference with the screen, the emotional andfinancial losses sustained by the worker can be extremely detrimental.The company's morale and financial well-being are also harmed. It is,therefore, very important to be able to identify a sample that willproduce a false positive.

[0084] Specifications for running urine samples vary from instrument toinstrument. Listed below is an example of parameters for the Hitachi 700series analyzer. The settings are intended as guidelines, and are setforth with the understanding that all those skilled in the art wouldrecognize that such parameters will vary from instrument to instrument.The suggested specifications for the Hitachi 700 series are as follows:Parameter settings for the Hitachi 700 series Test: [BR] Assay code: [1POINT] [50] - [0] Sample volume: [7] [5] Ri volume [210] [100] [NO] R2volume [ 0] [100] [NO] Wavelength [0] [660] Calib. Method: [Linear] [0][0] Std. (1) Conc.-POS: [0.0] * - [1]* assigned calibrator value Std.(2) Conc.-POS: [1.0] [2]assigned calibrator value Std. (3) Conc.-POS: []- [ ] Std. (4) Conc.-POS: [ ]- [ ] Std. (5) Conc.-POS: [ ]- [ ] Std.(6) Conc.-POS: [ ]- [ ] SD Limit: [999] Duplicate Limit: [32000]Sensitivity Limit: [0] ABS. Limit (INC/DEC): [32000] [INCREASE] ProzoneLimit: [0 ] [lower] Expected Value: [0.0] - [1.0] Tech. Limit: [0] -[1000] Instrument Factor [1.0]

[0085] Thus as described above, an unknown urine submitted for drugs ofabuse testing with no bromine present, will produce a value of less thanthe 1.0% Bromine calibrator. Conversely, if the sample has aconcentration of greater than 1.0% Bromine then it is an unacceptablesample and will result in false negative drug test results. Furthertesting can determine which of the above causes have produced theunacceptable specimen, however, it is irrelevant to the overall task athand, which is to determine the suitability of a specimen for drugs ofabuse testing.

[0086] To summarize more specifically Example 2, the automated methodfor the detection of adulteration of an unknown sample of urinesubmitted for drugs of abuse immunoassay testing comprising the steps ofplacing aliquots of an unknown urine (or other biological sample i.e.serum, whole blood, cerebral spinal fluid, gastric fluid, hairhomogenates, sweat extracts, saliva or other biological fluid) andcalibrator to be tested in automated analyzer sampling cups, placing thecups in a sampling tray within an automated analyzer, transferring thealiquots of sample and calibrator to cuvettes mounted within theautomated analyzer, injecting a first reagent composition (R-1)comprising an indicator and buffer in an aqueous medium into thecuvettes, mixing sample and reagent, and mixing sample and reagents, andreading absorbance values of reaction mixture composed of reagents andtest samples (said test samples include urine specimens, controls, andcalibrator) at specified intervals, in accordance with a preprogrammedcode introduced into the automated analyzer, at a preprogrammedmonochromatically specified wavelength, and comparing absorbance of thefirst reagent composition plus the unknown samples with that of thefirst reagent composition plus the calibrator containing a zeroreference point (normal urinary matrix), and thereby determining thepresence or absence of oxidants which adversely affects the reactionkinetics of the DAU assay being used by the drug testing laboratory.

[0087] The following changes to the above reagent solutions will remainwithin the scope and function of this invention and will have similarresults to the example above. The indicator in the solution 1,1,3-benzenediamine, could be substituted with one or more of thefollowing compounds including could be substituted with one or more ofthe following compounds including 1,2,3,4-tetrahydrobenzo(h)quinolone,1,2,3,4-tetrahydrobenzo(h)quinaldine,3-hydroxy-1,2,3,4-tetrahydrobenzo(h)quinolone,3-hydroxy-N-methyl-1,2,3,4-tetrahydrobenzo(h)quinolone,3-acetoxy-N-methyl-1,2,3,4-tetrahydrobenzo(h)quinolone,N-methyl-1,2,3,4-tetrahydrobenzo(h)quinolone,1,2,3,4-tetrahydroquinoline, 1,2,3,4-tetrahydroisoquinolinehydrochloride, 7,8-benzoquinoline,1,2,3,4-tetrahydro-3-isoquinolinecarboxylicacid hydrochloride,1,2,3,4-tetrahydro-1-napthylamine hydrochloride, napthylamine,N,N-dialkyl-alpha-napthylamine, phenolphthalin,2,2′-Azino-di-(3-ethylbenzthiazolinesulfonic acid),2,2′-Azino-di-(3-ethylbenzthiazolinesulfonic acid),2′-Azino-di-(3-ethylbenzthiazolinesulfonic acid) diammonium salt,cyanoditoly tetrazolium chloride, 3,3′-diaminobenzidine, o-dianisidine,dimethoxybenzidine, m-phenylenediamine, 3-amino-9-ethylcarbazole,3,3′-5,5′-tetramethylbenzidine, dimethoxybenzidine, 8-hydroxyquinoline,o-phenylenediamine, 3-dimethylaminobenzoic acid, 5-aminosalicylic acid,and 4-chloro-1-napthol, and another indicator that is suitable for thepresent device is 4-aminoantipyrine in combination with one of thefollowing compounds; p-hydroxybenzene sulfonate, p-hydroxybenzoic acid,n-ethyl-n-(2-hydroxy-3-sulfopropyl)-m-toluidine,n-ethyl-n-sulfopropyl-m-toluidine, 2-hydroxy-3,5-dichlorobenzenesulfonicacid, 3-hydroxy-2,4,6-triiodobenzoic acid, and3-hydroxy-2,4,6-tribromobenzoic acid. In addition, some of the aboveindicators require a diazotizable amine in the form of sulfanilic acid,arsanilic acid, sulfanilamide, aminobenzoic acid, or other suitableamine.

[0088] The hydrochloric acid buffer in solution 1, may be substitutedwith one or more of the following buffers: citrate, borate, borax,sodium tetraborate decahydrate, sodium perchlorate, sodium chlorate,sodium carbonate, MES (2-[N-Morpholino]ethanesulfonic acid),(Tris[hydroxymethyl]aminomethane), BIS-TRIS(bis[2-Hydroxyethyl]iminotris[hydroxymethyl]methane;2-bis[2-Hydroxyethyl]amino-2-[hydroxymethyl-1,3-propanediol), ADA(N-[2-Acetamidol]-2-iminodiacetic acid; N-[Carbaoylmethyl]iminodiacetcacid), ACES (2-[(2-Amino-2-oxoethyl)amino]ethanesulfonic acid;N-[2-Acetamido]-2-aminoethanesulfonic acid), PIPES(PiperazineN-N′-bis[2-ethanesulfonic acid)]; 1,4-Piperzinedethanesulfoicacid), MOPSO (3-[N-Morpholinol]-2-hydroxypropanesulfonic acid), BIS-TRISPROPANE (1,3-bis[tris(Hydroxymethyl)methylamino]propane), BES(N,N-bis[2-Hydroxyethyl]-2-aminoethaesulfonic acid;2-bis(2-Hydroxyethyl)amino]ethanesulfonic acid), MOPS(3-[N-Morpholino]propanesulfonic acid), TES(N-tris[Hydroxymethyl]methyl-2-aminomethanesulfonic acid;2[2-Hysroxy-1,1-bis(hydroxymethyl)-ethyl]amino)ethanesulfonic acid),HEPES (N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]), DIPSO(3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesulfonic acid), TAPSO(3-[N-tris(Hydroxyethyl)methylamino]-2-hydroxypropanesulfonic acid),HEPPSO (N-[2-Hydroxythyl]piperazine-N′-[2Hydroxypropanesulfonic acid]),POPSO (Piperazine-N,N′-bis[2-hydroxypropanesulfonic acid]), EPPS(N-[2-Hydroxyethyl]piperazine-N′-[3-propanesulfonic acid), TEA(triethanolamine), TRICINE (N-tris[Hydroxymethyl]methyllycine;N-[2-Hydroxy-1-1-bis(hydroxymethyl)etyyl]glycine), BICINE(N,N-bis[2-Hydroxyethyl]glycine), TAPS(N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid;([2-Hdroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-propanesulfonic acid),AMPSO (3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonicacid), CHES (2-[N-Cyclohexylamino]ethanesulfonic acid), CAPSO(3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid), AMP2-Amino-2-ethyl-1-propanol, CAPS (3-[cyclohexylamino]-1-propanesulfonicacid), hydrochloric acid, phosphoric acid, lactic acid, sulfuric acid,nitric acid, chromic acid, boric acid, perchloric acid, potassiumhydrogen tartrate, potassium hydrogen phthalate, calcium hydroxide,phosphate, bicarbonate, sodium hydroxide, potassium hydroxide, oxalateor succinate. Other buffers with an effective pK and pH range, andcapacity suitable for maintaining the sample-reagent mixture within therequired parameters of the assay's reaction mechanism may be added tothe above group, however acidic buffers are preferred.

EXAMPLE 3

[0089] This example will illustrate in detail the exact method formanufacturing the lateral flow bromine method. Keep in mind this methodcould be utilized for any general chemistry “test pad” or pads that arecurrently used or will be used in the art. In the case of DLFHtechnology, the manufacturing process includes impregnating onto anabsorbent, solid carrier (e.g. paper) called in this example, the “testpad”, in exactly the same manner as Example 1 with similar constituents.The test pad, once impregnated, is dried, then mounted onto a solidsupport (nitrocellulose membrane) that is capable of transporting(through lateral flow) liquid to the test pad from the point ofapplication of a test sample. In simpler terms, the device is dippedinto a liquid or the liquid sample is placed on the device at the bottomor starting point for the assay. The liquid migrates from the startingapplication point to the opposite end of the nitrocellulose lateral flowpaper, during which the test pad becomes saturated with the sample. Thereaction takes place on the test pad and color develops. The developedcolor is then compared to a color chart with known concentrations ofbromine that has the appropriate colors relative to each specificconcentration of bromine(s). The results are then recorded. Note, thetest pad must be an absorbent (wicking) material that permits migrationof sample up the solid absorbent test pad and allows analytes andreactants to interact.

[0090] Absorbent material is successively impregnated with the followingsolutions and dried at 25 degree C.:

[0091] Solution 1

[0092] napthylamine 2.0 g

[0093] aminobenzoic acid 6.0 g

[0094] oxalic acid 12.6 g

[0095] pH solution to a pH of 2.0

[0096] lab notes: the pH of the 1 liter solution should be acidic

[0097] In this example, the lateral flow device is prepared inaccordance with the instant invention. The lateral flow device iscomprised of a paper carrier matrix (S&S, 593 grade filter paper)impregnated with the compositions of solution 1. The paper is then cutinto test pads 5 mm by 5 mm. Note that said concentrations of any of theabove constituents can be varied to suit the DLFH lateral flow/dipstickdevice format (e.g. dependent upon paper type, and inclusion ofsemi-permeable membranes or other innovations utilized in dry chemistrytechnology). The paper is then dried using forced air. The driedimpregnated test pad is then placed at approximately 35 mm (in themiddle) of a 5 mm wide by 70 mm long nitrocellulose membrane (S&SFastTrack™ NC) and makes fluid contact with nitrocellulose lateral flowpaper. The nitrocellulose membrane is capable of transporting a liquidby capillary action or wicking from one end of the lateral flow deviceto the other in approximately 60 seconds. In this example, the DLFH hasthe dimensions of 5 mm wide by 70 mm long and can be backed by or incontact with strips of glass fiber filter material (e.g. S&S 30 grade)to aid in controlling the wicking action, or other solid supportmaterial can be used.

[0098] Again, to completely illustrate the present device the startingpoint or origin at which the sample is placed on the test device is 5 mmfrom one end of the strip, and 35 mm from the site of where the test padis placed in fluid contact with the strip. For simplicity, this examplewill have the 5 mm by 5 mm impregnated test pad placed on top of thelateral flow paper and thus be in fluid contact with the said paper.

[0099] The mechanics of how the present art's LFD and dipstick test padhybrid may be explained is as follows. The starting point or origin atwhich the sample is placed on the test device is 5 mm from one end ofthe strip, and 35 mm from site where the chemically impregnated test padis in fluid contact with the lateral flow paper. The test pad can beplaced on top of the lateral flow paper making fluid contact with thelateral flow paper from the bottom side of the test pad, or the lateralflow paper can touch the paper from the side of the test pad and remainin fluid contact with the test pad. Or the lateral flow paper can reston top of the edge of test pad or be attached and in fluid contact withthe test pad in some other manner. One of the novel advantages in usinga hybrid device made of lateral flow material and a dry chemistry testpad is the lack of cross contamination from one pad to the next fromexcessive fluid, as is inherent in the prior art. For illustration,currently there are available many different types of dry chemistry teststrips available, such as the Miles Laboratories, Inc. MULTISTIXref.This device and many other like it has multiple reagents test pads withdifferent chemistries impregnated onto each pad on a single supportmembrane backing (usually plastic). Because of the relative proximity ofthese pads to each other on the same device it is easy for crosscontamination to occur, causing unreliable results. This is called“runover” (i.e. when a reagent from one pad runs over another adjacenttest pad). The present arts eliminates runover. The applicants novelapproach to the solution of runover has not been taught prior to thepresent art and is the result of extensive research and development.

[0100] Result interpretation can be explained as follows. If the sampleis positive, with a concentration of 1.0 mg/dL bromine or more, thefollowing occurs. A drop of urine (approximately 50 uL) is applied atthe starting point or origin of the strip. The urine then migrates tothe opposite or terminal end of the strip. As the urine migrates acrossthe lateral flow material (nitrocellulose) and comes into contact withthe test pad (filter paper), the urine will saturate the pad and cause achemical reaction between the impregnated chemicals and bromine(s) inthe urine. A blue to purple color will develop on the test padindicating a positive (greater than 1.0 mg/dL bromine) for the presenceof bromine. This color can then be compared to a color chart showing thedifferent colors from colorless (white background)) to a dark blue topurple depending upon the concentration of the bromine(s), if greaterthan 1.0 mg/dL. The reaction on the test pad is immediate thus the testresults can be observed immediately.

[0101] If the sample is negative, with a concentration of less than 1.0mg/dL of bromine present the following occurs. A drop of urine(approximately 50 uL) is applied at the starting point or origin of thestrip. The urine then migrates to the opposite or terminal end of thestrip. As the urine migrates across the lateral flow material and comesinto contact with the test pad, the urine will saturate the pad andcause a chemical reaction between the impregnated chemicals and bromine.However, this example is for a negative result, thus, no reaction occursand no color develops, indicating a negative result. This negativeresult color can then be compared to a color chart showing the differentcolors from no color developed (negative) to dark blue to purpledepending upon the concentration of the bromine(s), if greater than 1.0mg/dL. The reaction on the test pad is immediate thus the test resultscan be observed immediately.

[0102] Changes to the above reagent solution of example 3 can be madeand still remain within the scope and function of this invention andwill have similar results to examples 1 and 2 above. The indicator(s)and buffer(s) of example 3 can be replaced by all the examples andpossible substitutions as illustrated in example 1.

[0103] This brief description of the present art illustrates acompletely enabled device that would allow a physician, patient, and/ortechnician to quickly and easily determine the presence of the brominein urine, providing a much needed advancement in the art of adulterationtesting.

[0104] To briefly explain the present device as taught. The present artincludes a device for the detection of bromine adulteration in a sampleof urine submitted for drugs of abuse immunoassay testing the stepscomprise of preparing a dry chemistry test means by successivelyimpregnating a solid, carrier matrix with reagent solutions containingan indicator and a buffer, and drying the impregnated, solid carriermatrix. Finally, by dipping said dry chemistry test means into urine,one can observe the detectable response in the form of a color developedin the presence or absence of bromine. This present art also illustratesa unique device that will prevent cross contamination (runover) of testpads on the same dipstick, as well as a unique dry chemistry test padlateral flow device hybrid. These methods can incorporate detectableresponses in the visible color range to the human eye or in the visiblelight spectrum. In addition the present art can be used for clinicaldiagnostics of bromine toxicity. These methods have a wide sample choiceother than urine, and can be replaced by any biological sample includingserum, whole blood, cerebral spinal fluid, gastric fluid, hairhomogenates, sweat extracts, saliva or other biological fluid.

EXAMPLE 4

[0105] This is a method for manufacturing a dry chemistry dipstick (teststrip) with a solid carrier for the adulteration detection of oxidantsin urine samples submitted for drugs of abuse analysis. Filter paper issuccessively impregnated with the following solutions and dried at 25degree C.:

[0106] Solution I

[0107] bis[2-Hydroxyethyl]iminotris[hydroxymethyl]methane 20.9 g

[0108] distilled water added to 100 mL total volume of solution

[0109] pH the solution to a value between 1.0 and 12.5 preferably 5.0

[0110] page 38

[0111] lab notes: Buffer strength is preferably 0.01 Molar or greater

[0112] Solution 2

[0113] m-Phenylenediamine (MPD) 0.01 g/L

[0114] distilled water added to make 1000 mL total volume of solution

[0115] lab notes: MPD needs to be in solution at a concentration 0.001g/L or greater

[0116] In this example a dipstick was prepared in accordance with theinstant invention. The device comprised a paper carrier or solid matrixincorporated with the composition of solutions 1 and 2 above. Note thatthe concentrations of any of the following examples can be varied tosuit the dipstick device format (dependent upon paper type, or use ofsemi-permeable membrane or other suitable material). This example iscarried out using the following procedure. To produce the test means, apiece of Whatman 3 MM filter paper having approximate dimensions of 1inch by 3 inches was impregnated with solution 1 by immersing the paperinto solution 1. The paper was then dried by using forced air notexceeding 25° C. A second piece of Whatman 1 MM filter paper havingapproximate dimensions of 1 inch by 3 inches was impregnated withsolution 2 by immersing the paper into solution 2. The paper was thendried by using forced air not exceeding 25° C. The dried papers are thenlaminated to each other by the use of a non-reactive (neutral adhesive).The dried, laminated papers are then applied to one side of a piece ofdouble-sided adhesive transfer tape commercially available from 3MCompany, St. Paul, Minn. 55144. The laminate is then slit into portionsmeasuring 3 inches by 0.2 inches. One portion is then attached, via theunused adhesive side to a polystyrene sheet measuring about 1.5 inchesby 3 inches and the resulting laminate is slit parallel to its shortdimension to form test devices comprising a 1.5 inch oblong polystyrenestrip carrying a square of the impregnated papers at one end, the otherend serving as the handle. When the dipstick thus obtained is dippedinto a urine submitted for drugs of abuse testing, and no uniform bluishcolor develops then no bromine(s) is present. Conversely, if anyconcentration of bromine(s) is present in the urine at a 0.1% v/v orgreater a bluish color will develop thus confirming the presence ofbromine(s).

[0117] In summary, Example 4 is as follows: the foregoing dry chemistrytest strip (dipstick) method for the adulteration detection of brominecontaining adulterants in a sample of urine submitted for drugs of abuseimmunoassay testing comprises the steps of preparing a test means bysuccessively impregnating a solid, carrier matrix with reagentsolutions, drying the impregnated, solid test means, then dipping saiddried test means into urine, and finally observing any color change inthe presence or absence of bromine(s).

[0118] Changes to the above reagent solution of example 4 can be madeand still remain within the scope and function of this invention andwill have similar results to examples 1, 2 and 3 above. The indicator(s)and buffer(s) of example 4 can be replaced by all the examples andpossible substitutions as illustrated in example 1.

[0119] This brief description of the present art illustrates acompletely enabled device that would allow a physician, patient, and/ortechnician to quickly and easily determine the presence of an adulterantthat contains bromine(s) in urine, providing a much needed advancementin the art of adulteration testing. The same devices as illustratedcould be used for clinical diagnostic, industrial or other purposesusing the same means.

[0120] To briefly explain the present device as taught. The present artincludes a device for the detection of bromine or analogs of bromine (orother states of bromine such as sodium bromate, potassium bromate, etc.)in a sample of urine comprising the steps of preparing a dry chemistrytest means by successively impregnating a solid, carrier matrix withreagent solutions containing an indicator and a buffer, and drying theimpregnated, solid carrier matrix. Finally, by dipping said drychemistry test means into urine, one can observe the detectable responsein the form of a color developed in the presence or absence of bromine.This present art also illustrates a unique device that will preventcross contamination (runover) of test pads on the same dipstick, as wellas a unique dry chemistry test pad lateral flow device hybrid. Thesemethods can incorporate detectable responses in the visible color rangeto the human eye or in the visible light spectrum. These methods have awide sample choice other than urine, and can be replaced by anybiological sample including serum, whole blood, cerebral spinal fluid,gastric fluid, hair homogenates, sweat extracts, saliva or otherbiological fluid.

[0121] It is understood paste that remarkable discovery of the new artformula and indicator(s) for bromine in the form of bromine in urine (aswell as the other matrices mentioned) that was unknown prior to thisart. The newly discovered and suitable indicators for the present artformula are one or more of the following;1,2,3,4-tetrahydrobenzo(h)quinolin-3-ol,1,2,3,4-tetrahydrobenzo(h)quinolone,1,2,3,4-tetrahydrobenzo(h)quinaldine,3-hydroxy-1,2,3,4-tetrahydrobenzo(h)quinolone,3-hydroxy-N-methyl-1,2,3,4-tetrahydrobenzo(h)quinolone,3-acetoxy-N-methyl-1,2,3,4-tetrahydrobenzo(h)quinolone,N-methyl-1,2,3,4-tetrahydrobenzo(h)quinolone, 1,3-phenylenediamine,1,2,3,4-tetrahydroquinoline, 1,2,3,4-tetrahydroisoquinolinehydrochloride, 7,8-benzoquinoline,1,2,3,4-tetrahydro-3-isoquinolinecarboxylicacid hydrochloride,1,2,3,4-tetrahydro-1-napthylamine hydrochloride, napthylamine,N,N-dialkyl-alpha-napthylamine, phenolphthalin,2,2′-Azino-di-(3-ethylbenzthiazolinesulfonic acid),2,2′-Azino-di-(3-ethylbenzthiazolinesulfonic acid) diammonium salt,cyanoditoly tetrazolium chloride, 3,3′-diaminobenzidine, o-dianisidine,dimethoxybenzidine, 0-phenylenediamine, 3-amino-9-ethylcarbazole,3,3′-5,5′-tetramethylbenzidine, dimethoxybenzidine, 8-hydroxyquinoline,m-phenylenediamine, 3-dimethylaminobenzoic acid, 5-aminosalicylic acid,and 4-chloro-1-napthol, and another indicator that is suitable for thepresent device is 4-aminoantipyrine in combination with one of thefollowing compounds; p-hydroxybenzene sulfonate, p-hydroxybenzoic acid,n-ethyl-n-(2-hydroxy-3-sulfopropyl)-m-toluidine,n-ethyl-n-sulfopropyl-m-toluidine, 2-hydroxy-3,5-dichlorobenzenesulfonicacid, 3-hydroxy-2,4,6-triiodobenzoic acid, and3-hydroxy-2,4,6-tribromobenzoic acid. In addition, some of the aboveindicators require a diazotizable amine in the form of sulfanilic acid,arsanilic acid, sulfanilamide, aminobenzoic acid, or other suitableamine. It is understood that the present arts discovery of the use ofindicators such as the ones mentioned above or others that have not beenmentioned that are sensitive to bromine oxidation-reduction reactions inbiological matrices such as urine and are capable of producing adetectable response in the presence of a bromine oxidation-reductionreaction are within the present art. Therefore the use of a bromineoxidation-reduction indicator that is not mentioned here would fallwithin the spirit and scope of the present invention.

[0122] The subject invention, utilizing this concept, provides anextraordinary means for determining adulteration in a urine or otherbiological specimen submitted for drugs of abuse immunoassay with asingle assay for bromine(s). Furthermore, the subject invention willdiscourage additional attempts to use bromine(s) (or any form ofbromine) as adulterants in samples to be tested via immunoassay systems.

[0123] In addition, and most importantly the present art provides asingle test means that allows for the detection of all bromine formsthat may be used as adulterants such as potassium bromate and sodiumbromate to name a few with a single assay. This unique and markedadvance in the art of adulteration will have an immediate beneficialimpact on the drug and adulteration testing industry.

[0124] It is understood that variations or modifications in thefollowing embodiments may be made by someone skilled in the art withoutdeparting from the spirit and scope of the invention. All suchmodifications and variations are to be included within the scope of theinvention as defined in the appended claims:

I claim:
 1. The method for the detection of bromine in a biologicalsample, wherein the biological sample is selected from the groupconsisting essentially of urine, serum, whole blood, cerebral spinalfluid, gastric fluid, hair homogenates, sweat extracts or saliva,consisting essentially of; (A) preparing a liquid chemistry test meanswith reagent solutions consisting essentially of an indicator and abuffer, wherein the indicator is selected from the group consisting of1,2,3,4-tetrahydrobenzo(h)quinolin-3-ol,1,2,3,4-tetrahydrobenzo(h)quinolone,1,2,3,4-tetrahydrobenzo(h)quinaldine,3-hydroxy-1,2,3,4-tetrahydrobenzo(h)quinolone,3-hydroxy-N-methyl-1,2,3,4-tetrahydrobenzo(h)quinolone,3-acetoxy-N-methyl-1,2,3,4-tetrahydrobenzo(h)quinolone,N-methyl-1,2,3,4-tetrahydrobenzo(h)quinolone, 1,3-phenylenediamine,1,2,3,4-tetrahydroquinoline, 1,2,3,4-tetrahydroisoquinolinehydrochloride, 7,8-benzoquinoline,1,2,3,4-tetrahydro-3-isoquinolinecarboxylicacid hydrochloride,1,2,3,4-tetrahydro-1-napthylamine hydrochloride, napthylamine,N,N-dialkyl-alpha-napthylamine, phenolphthalin,2,2′-Azino-di-(3-ethylbenzthiazolinesulfonic acid),2,2′-Azino-di-(3-ethylbenzthiazolinesulfonic acid) diammonium salt,cyanoditoly tetrazolium chloride, 3,3′-diaminobenzidine, o-dianisidine,dimethoxybenzidine, 0-phenylenediamine, 3-amino-9-ethylcarbazole,3,3′-5,5′-tetramethylbenzidine, dimethoxybenzidine, 8-hydroxyquinoline,m-phenylenediamine, 3-dimethylaminobenzoic acid, 5-aminosalicylic acid,4-chloro-1-napthol, diazotizable amine, sulfanilic acid, arsanilic acid,sulfanilamide, aminobenzoic acid or 4-aminoantipyrine in combinationwith one of the following compounds; p-hydroxybenzene sulfonate,p-hydroxybenzoic acid, n-ethyl-n-(2-hydroxy-3-sulfopropyl)-m-toluidine,n-ethyl-n-sulfopropyl-m-toluidine, 2-hydroxy-3,5-dichlorobenzenesulfonicacid, 3-hydroxy-2,4,6-triiodobenzoic acid, and3-hydroxy-2,4,6-tribromobenzoic acid; (B) placing the reagent in thereagent compartment of a chemistry autoanalyzer, aliquoting samples,calibrators, and controls into sample cups and placing them on thechemistry autoanalyzer; (C) and transferring aliquots of each sample,calibrator, and control into single discrete cuvettes mounted within thechemistry autoanalyzer; (D) aliquoting the said reagent into eachcuvette and mixing; and (E) incubating the reaction mixture; (F)measuring and recording absorbance values of the reaction mixtures withthe chemistry autoanalyzer's spectrophotometer at 340 nm to 800 nm atpreprogrammed time intervals; and (G) comparing absorbance values ofsamples and controls to those of calibrators in the form of a standardcurve thereby quantitating the bromine present.
 2. The method for thedetection of bromine in a biological sample, wherein the biologicalsample is selected from the group consisting essentially of urine,serum, whole blood, cerebral spinal fluid, gastric fluid, hairhomogenates, sweat extracts or saliva, consisting essentially of; (A)preparing a liquid chemistry test means with reagent solutionsconsisting essentially of an indicator and a buffer, wherein the bufferis selected from the group consisting of citrate, borate, borax, sodiumtetraborate decahydrate, sodium perchlorate, sodium chlorate, sodiumcarbonate, (Tris[hydroxymethyl]aminomethane),(2-[N-Morpholino]ethanesulfonic acid),(bis[2-Hydroxyethyl]iminotris[hydroxymethyl]methane;2-bis[2-hydroxyethyl]amino-2-[hydroxymethyl-1,3-propanediol),(N-[2-Acetamidol]-2-iminodiacetic acid; N-[Carbaoylmethyl]iminodiacetcacid), (2-[(2-Amino-2-oxoethyl)amino]ethanesulfonic acid;N-[2-Acetamido]-2-aminoethanesulfonic acid),(PiperazineN-N′-bis[2-ethanesulfonic acid)]; 1,4-Piperzinedethanesulfoicacid), (3-[N-Morpholinol]-2-hydroxypropanesulfonic acid),(1,3-bis[tris(Hydroxymethyl)methylamino]propane),(N,N-bis[2-Hydroxyethyl]-2-aminoethaesulfonic acid;2-bis(2-Hydroxyethyl)amino]ethanesulfonic acid),(3-[N-Morpholino]propanesulfonic acid),(N-tris[Hydroxymethyl]methyl-2-aminomethanesulfonic acid;2[2-Hysroxy-1,1-bis(hydroxymethyl)-ethyl]amino)ethanesulfonic acid),(3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesulfonic acid),(3-[N-tris(Hydroxyethyl)methylamino]-2-(hydroxypropanesulfonic acid),(N-[2-Hydroxythyl]piperazine-N′-[2Hydroxypropanesulfonic acid]),(Piperazine-N,N′-bis[2-hydroxypropanesulfonic acid]),(N-[2-Hydroxyethyl]piperazine-N′-[3-propanesulfonic acid),(triethanolamine), (N-tris[Hydroxymethyl]methyllycine;N-[2-Hydroxy-1-1-bis(hydroxymethyl)etyyl]glycine),(N,N-bis[2-Hydroxyethyl]glycine),(N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid;([2-Hdroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-propanesulfonic acid),(3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid),(2-[N-Cyclohexylamino]ethanesulfonic acid),(3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid),2-Amino-2-ethyl-1-propanol, (3-[cyclohexylamino]-1-propanesulfonicacid), hydrochloric acid, phosphoric acid, lactic acid, sulfuric acid,nitric acid, chromic acid, boric acid, citric acid, oxalic acid,tartaric acid, succinic acid, perchloric acid, potassium hydrogentartrate, potassium hydrogen phthalate, calcium hydroxide, phosphate,bicarbonate, sodium hydroxide, potassium hydroxide, tartrate, oxalate orsuccinate; (B) placing the reagent in the reagent compartment of achemistry autoanalyzer, aliquoting samples, calibrators, and controlsinto sample cups and placing them on the chemistry autoanalyzer; (C) andtransferring aliquots of each sample, calibrator, and control intosingle discrete cuvettes mounted within the chemistry autoanalyzer; (D)aliquoting the said reagent into each cuvette and mixing; and (E)incubating the reaction mixture; (F) measuring and recording absorbancevalues of the reaction mixtures with the chemistry autoanalyzer'sspectrophotometer at 340 nm to 800 nm at preprogrammed time intervals;and (G) comparing absorbance values of samples and controls to those ofcalibrators in the form of a standard curve thereby quantitating thebromine present.